ABSTRACT Glutamate is the primary neurotransmitter in the brain, and is important for the majority of synaptic transmission and therefore learning and memory formation. At glutamatergic synapses, glutamate is released from the axon of the first neuron into the synaptic cleft, eventually activating glutamate receptors on the postsynaptic membrane of an adjacent neuron. A family of secondary active glutamate transporters localized to neuronal and glial membranes quickly import excess glutamate back into the cell, through coupling to electrochemical gradients of sodium ions, potassium ions, and protons. Mutations in these genes cause excitotoxicity and are associated with neurological disorders including Amyotrophic Lateral Sclerosis, Alzheimer's disease, stroke, and epilepsy; however, structural and mechanistic characterization of the ion specificity of mammalian transporters has proved to be challenging, ultimately hindering new strategies of pharmacological modulation. The majority of our understanding of this family arose from studies of conserved bacterial glutamate transporters, which generally couple transport to either exclusively sodium or protons. The proposed studies will use evolutionary analysis with structural biology to determine the molecular basis of ion specificity in glutamate transporters. We will accomplish this goal by pursuing two specific aims: (1) Characterize the evolutionary switch to a proton-coupled transporter; and (2) Determine the ion specificity of an ancestral glutamate transporter predating divergence of Na+ and proton-coupled transporters. Successful completion of these studies will increase understanding of how homologous transporters can harness different ionic gradients.